Wild apple polyphenol and process for producing the same

ABSTRACT

A fruit polyphenol obtained by subjecting a crab apple fruit to pressing and/or extraction. The fruit polyphenol is rich in such components as condensed tannins (procyanidins), chlorogenic acid, and epicatechin. The fruit polyphenol can be produced economically and efficiently.

TECHNICAL FIELD

[0001] The present invention relates to a fruit polyphenol derived froma crab apple fruit, out of polyphenols known to have a variety offunctionalities, and a production process thereof.

BACKGROUND ART

[0002] A wide variety of polyphenols known as secondary metabolites ofplants are present in the plant kingdom. Some of these polyphenols havebeen found to show diversified physiological activities. It hasheretofore been disclosed that catechin contained in tea hasphysiological activities such as antibacterial activity, antioxidativeactivity, anticancerous activity, antiallergic activity, deodoringactivity, antiviral activity and blood cholesterol oxidation inhibitingactivity (See JP-A-63-214183, JP-A-2-6499, JP-A-4-178320, etc.).

[0003] Further, in JP-A-7-285876, it is reported that polyphenolscontained in extracts of unripe apples, unripe pears or unripe peacheshave activities such as antioxidative activity, blood pressure reducingactivity, antimutagenic activity, antiallergic activity, anticariogenicactivity and deodoring activity. In addition, in “Japanese and ChineseMedicine—New Edition” (author: Kaneyoshi Akamatsu, Ishiyaku SyuppanKabushiki Kaisha, published in 1980, pp. 359 to 360, J. LiquidChromatography, 15(4), 637 to 646(1992)), it is mentioned that applescontain tannins. However, all the mention is for apples to be eatenfresh or apples to be processed, and no mention is made of crab apples.

[0004] Thus, it has been found that tea and unripe fruits, for example,have diversified physiological activities. The present inventor has madeintensive studies on a process for producing a polyphenol having a widevariety of physiological activities economically and efficiently and onraw materials. As a result, the inventor has found that crab applescontain a significantly large amount of polyphenols, particularly acondensation tannin (procyanidin). The present invention has beencompleted based on this finding.

DISCLOSURE OF THE INVENTION

[0005] That is, according to the present invention, a fruit polyphenolis provided which is obtained by subjecting a crab apple fruit topressing and/or extraction.

[0006] Further, according to the present invention, a process forproducing a fruit polyphenol is provided which comprises the steps ofsubjecting a crab apple fruit to pressing and/or extraction and thenpurifying the resulting juice and/or extract so as to obtain apolyphenol fraction.

[0007] In the present invention, it is preferable to add sulfurous acidin a concentration of 350 to 3,000 ppm at the time of pressing a crabapple fruit. Further, when a crab apple fruit is subjected toextraction, it is preferable to add sulfurous acid in a concentration of100 ppm or higher if a proper amount of alcohol is mixed into the crabapple fruit which is then crushed. Then, after a clear extract isprepared, sulfurous acid is preferably added in a concentration of 50ppm or higher so as to prevent oxidation.

[0008] Best Mode for Carrying out the Invention

[0009] Hereinafter, the present invention will be described in detail.

[0010] A fruit polyphenol in the present invention comprises apolyphenol fraction obtained by subjecting a crab apple fruit topressing and/or extraction and then purifying the obtained juice orextract. The purification to obtain the polyphenol fraction is carriedout by treating the juice or extract with an adsorbent, and a fractionadsorbed to the adsorbent (hereinafter referred to as “adsorbedfraction”) contains a polyphenol. Then, the adsorbed fraction is elutedwith an anhydrous alcohol (such as ethanol) so as to obtain a purifiedpolyphenol fraction.

[0011] This polyphenol fraction can be further concentrated to obtain aliquid preparation. Further, when the concentrated polyphenol fractionis subjected to spray-drying or freeze-drying, a powdery preparation canbe obtained.

[0012] As a raw material of the polyphenol of the present invention, acrab apple fruit is used. An apple is a plant belonging to Malus ofRosaceae, and a crab apple refers to a species which has not beensubjected to breeding by a human or a species which is not or cannot beeaten fresh or processed. Further, as the raw material of the polyphenolof the present invention, a ripe fruit and an unripe fruit can be used.

[0013] Illustrative examples of species of crab apples include Hu BeiHai Tang, Yin Gui Hai Tang, Xi Fu Hai Tang, Zhong Guo Ping Guo, Cui Guo,Ya Hai Tang, Ya Sha Guo and Ya Ping Guo which are native to China, AdamsCrab, Geneva, Gorgeous, Jay Darling, Eleyi, Eley Purple Crab, MalusRobusta, Makamic Crab, Profusion, Royalty, Liset Crab, Red SplendorCrab, Tar Tan, and Lemoine Purple Crab.

[0014] It is said that apples are originated in the Caucasus Provincenear the Caspian Sea. It is known that a vast number of crab apples havegrown in areas from the Uyghur Province in China to Kazakhstan andUzbekistan bordering the Caspian Sea since a long time ago.Particularly, in the vicinity of the Tian Shan which extends fromShin-gen and I-rei in the Uyghur Province in China to the eastern partof Kazakhstan, a large-scale old growth forest of crab apples spreads.Crab apples in this area have hardly been used by humans. The crab applecontains a large amount of preferable polyphenol component and cantherefore be suitably used in the present invention.

[0015] Further, also in Europe and North America, trees of crab appleswhich are apples without breeding are still observed in many places.These crab trees can also be suitably used in the present invention.

[0016] As a pressing method, there can be used a method which comprisescleaning an apple fruit as a raw material, crushing and pressing theapple fruit with or without addition of sulfurous acid so as to obtain ajuice, preferably adding a pectolytic enzyme, and then subjecting theresulting mixture to means such as centrifugation or filtration so as toobtain a clear juice. Meanwhile, as an extraction method, there can beused a method which comprises adding sulfurous acid to a cleaned rawmaterial as required, mixing the material with alcohol (such as ethanolor methanol), crushing the mixture, extracting the resulting materialwhile the material is being immersed and pressed or being refluxed underheating, concentrating the extract under a reduced pressure so as toremove the alcohol, and subjecting the concentrate to centrifugation andfiltration or to distribution with an organic solvent such as hexane orchloroform and filtration so as to obtain a clear extract.

[0017] As described above, in the present invention, sulfurous acid ispreferably added at the time of pressing and/or extraction. In general,when an apple is pressed to obtain a juice as described above, anappropriate antioxidant is added so as to prevent the juice from beingdiscolored brown by oxidation. Illustrative examples of conventionallyused antioxidants include sulfurous acid and vitamin C. A commonly usedconcentration of sulfurous acid is 200 ppm or lower. Further, to attainan antioxidative effect that is nearly the same as that of sulfurousacid, vitamin C must be added in a concentration of 1,000 ppm or higher.This is because the concentration of sulfurous acid used in productionof alcoholic beverages (such as fruit wine) is lower than 350 ppm whichis a maximum amount permitted to be added by the liquor tax law and itis generally not attempted to add sulfurous acid in a concentration of350 ppm or higher. When sulfurous acid is added in a concentration of350 ppm or higher, the obtained juice has a strong odor of sulfurousacid and cannot be drunk.

[0018] However, as a result of intensive studies made by the presentinventor, it has been found that when sulfurous acid is added in aconcentration of 350 to 3,000 ppm, more preferably 1,000 to 2,000 ppm,more specifically, when a sulfite such as potassium metabisulfite isadded in a concentration of 350 to 3,000 ppm, more preferably 1,000 to2,000 ppm in terms of sulfurous acid at the time of pressing an apple soas to obtain a juice, a polyphenol component which is intrinsicallycontained in the apple can be extracted without damaging the component.When the concentration of sulfurous acid is lower than the above range,the obtained juice undergoes an oxidation reaction, and the contents oflow-molecular-weight components [such as catechin, epicatechin,procyanidin B1 (PB1), procyanidin B2 (PB2) and procyanidin C1 (PC1)] aresignificantly lowered although the total content of all polyphenols inthe obtained juice does not change. Although details of the reaction areunknown, it is assumed that small molecule are polymerized and modified,because the total polyphenol content hardly changes.

[0019] As for vitamin C, when it is added in a concentration of 3,500 to30,000 ppm, more preferably 10,00 to 20,000 ppm, it can produce the sameeffect as that obtained by addition of sulfurous acid in the aboveconcentration range.

[0020] Meanwhile, when an extraction method is employed, sulfurous acidis preferably added in a concentration of 100 ppm or higher, when aproper amount of alcohol is mixed into above, after the alcohol isremoved and centrifugation and filtration or distribution and filtrationare carried out to obtain a clear extract, sulfurous acid is preferablyadded in a concentration of 50 ppm or higher so as to prevent oxidation.

[0021] As for a purification method, the above clear juice or clearextract is passed through a column filled with an adsorbent capable ofselectvely adsorbing a polyphenol and releasing the adsorbed phenol byuse of an eluant, such as a styrene-divinylbenzene type syntheticadsorption resin, an anion exchange resin or an octadecyl groupchemically bonded silica gel (ODS) so as to adsorb a polyphenolfraction. Then, water is passed through the column for washing. Then, a10-100% alochol (e.g. ethanol) solution, prefereably an about 50%alcohol solution is passed through the column, whereby the polyphenolfraction can be eluted and recovered. The obtained polyphenol solutionis concentrated under a reduced pressure so as to remove the alcohol,whereby a fruit polyphenol liquid preparation can be obtained. Theliquid preparation may contain an organic acid such as malic acid, afatty acid ester or the like. Further, the liquid preparation may bespray-drying or freeze-drying either as it is or after an auxiliaryagent for powdering such as dextrin or the like is added, whereby afruit polyphenol powdery preparation can be obtained.

[0022] The fruit polyphenol obtained in the present invention comprisescaffeic acid derivatives, p-coumaric acid derivatives, flavan-3-ols(catechins), flavonols (quercetin glycosides), dihydrochalcones(phloretin glycosides) and the like as simple polyphenol compounds andcondensed tannins (procyanidins) and the like as high-molecularpolyphenol compounds. A polyphenol obtained from a crab apple contains aparticularly large quantity of condensed tannins.

[0023] Further, crab apples of some type contain a specific polyphenolcomponent in particularly large quantity, and conversely, crab apples ofsome other type contain almost no specific polyphenol component. By useof these crab apples, purification of the specific polyphenol componentcan be carried out very efficiently.

[0024] The fruit polyphenol obtained in the present invention has avariety of physiological activities. The functionalities of a polyphenolderived from an apple are disclosed in JP-A-7-285876. The polyphenol inthe present invention has the activities described in the publication,i.e., antioxidative activity, blood pressure reducing activity,antimutagenic activity, antiallergic activity, anticariogenic activityand deodoring activity.

[0025] Therefore, the fruit polyphenol produced as described above canbe used in drugs as agents having antioxidative activity, blood pressurereducing activity, antimutagenic activity, antiallergic activity,anticariogenic activity and deodoring activity. Drugs containing thefruit polyphenol can be prepared by a known method in the form of oraldrugs such as a tablet, powders, granules, a capsule and a syrup orparenteral drugs such as a suppository, ointment, nebula and injection.

[0026] Further, the fruit polyphenol can be added to beverages and foodsin general so that they can be suitably used as beverages and foodshaving antioxidative activity, blood pressure reducing activity,antimutagenic activity, antiallergic activity, anticariogenic activityand deodoring activity. To be more specific, the fruit polyphenol can beadded to alcohol beverages, carbonated beverages, fruit beverages,lactic acid bacteria beverages, coffee, tea, ice cream, candies, chewinggums, snacks, breads, noodle, and the like.

[0027] Furthermore, the fruit polyphenol can also be added to cosmetics.Illustrative examples of cosmetics to which the fruit polyphenol can beadded include skin-care cosmetics and bath cosmetics such as soap,facial wash, cream, skin milk, skin lotions, powders, perfume andlipsticks; hair-care cosmetics such as shampoo and rinse; andtoothpaste.

[0028] Most of the various functionalities of the above fruit polyphenolaccording to the present invention are ascribable to procyanidinfractions. As described in the above description of the productionprocess, these fractions may be oxidized and modified during production.Thus, it is important to carry out a purification step in a short timewith the oxidation suppressed as much as possible. When a crab applecontaining a larger amount of procyanidin that an unripe apple fruit ora crab apple having composition leaning to a specific component is used,the production step can be simplified, and separation and purificationcan be carried out in a shorter time.

[0029] Further, in the case of unripe fruits of cultivated species suchas “Fuji”, apple fruits cultivated with special care must be collectedby thinning one by one carefully during their growth. Meanwhile, in thecase of crab apples, they grow naturally in the field, neither afertilizer nor a pesticide must be used for cultivation thereof, andneither efforts nor costs are required. Further, since no pesticide isused, the crab apples are high in the safety. In addition, uponharvesting of the crab apples, unlike thinning works of unripe fruits ofcultivated species, all fruits on a tree can be collected. Consequently,the harvesting work is quite easy.

[0030] Hereinafter, the present invention will be further described withreference to Examples. The present invention, however, shall not belimited by these Examples in any way.

EXAMPLE 1

[0031] [General Compositional Analysis of Crab Apple Juice]

[0032] Ripe crab apples were subjected to general compositionalanalysis.

[0033] Samples: Various ripe crab apples shown in Table 1 and a ripeapple “Fuji” as a comparison were used.

[0034] Production Process of Samples: The fruit samples were crushed ina mixer and pressed while 1,000 ppm in terms of sulfurous acid ofpotassium metabisulfite was being added thereto as an antioxidant. Theobtained juices were subjected to centrifugation and then filtration soas to obtain clear juices. The obtained clear juices were measured forthe following items, and the results of the measurements are shown inTable 1.

[0035] (Measurement Items)

[0036] Average Weight of Individual Fruit

[0037] Acidity (in terms of malic acid)

[0038] Brix

[0039] Concentration of Saccharose: Measured by high-performance liquidchromatography

[0040] Concentration of Glucose: Measured by high-performance liquidchromatography TABLE 1 Average Weight of Acidity Concen- Concen-Individual (in terms tration of tration of Fruit of malic SaccharoseGlucose Kind (g) acid, g/L) Brix (g/L) (g/L) Hu Bei Hai 6 19 15.7 45 28Tang Yin Gyi Hai 20 19 13.7 31 14 Tang Xi Fu Hai Tang 5 15 14.9 29 22Zhong Guo 143 22 11.0 9.7 23 Ping Guo Cui Guo 16 12 17.0 22 45 Fuji 2563.0 11.7 25 27

[0041] As shown in Table 1, the ripe crab apples were smaller than theripe apple fruit of cultivated species. Insofar as the general analysisvalues thus measured, none of them particularly characterized the crabapples.

EXAMPLE 2

[0042] (Analysis of Polyphenol in Crab Apple Juice)

[0043] Various kinds of crab apple juices were prepared in the samemanner as in the above Example 1. A total polyphenol content wasmeasured by a Folin-Ciocalteu method and expressed also in terms ofchlorogenic acid. Further, a total procyanidin content was measured by aPorter method and expressed also in terms of procyanidin B2. Otherpolyphenol components were measured quantitatively by high-performanceliquid chromatography. The results are shown in Tables 2 and 3. TABLE 2Total Polyphenol Chlorogenic Acid Epicatechin Total Procyanidin PB1 PB2PC1 Kind Content (ppm) (ppm) (ppm) Content (ppm) (ppm) (ppm) (ppm) HuBei Hai Tang 17442 104 517 13817 196 891 423 Yin Gyi Hai Tang 15678 731538 11669 27 983 460 Xi Fu Hai Tang 9361 142 176 6420 45 272 125 ZhongGuo Ping Guo 3933 141 191 1147 51 109 39 Cui Guo 11583 629 345 7560 40615 269 Adams Crab 23570 100 801 16827 81 1367 736 Geneva 6331 270 2712085 86 351 128 Gorgeous 5671 64 21 1514 trace 80 14 Jay Darling 9417325 484 6068 81 784 306 Eleyi 10466 566 558 9229 115 1023 365 EleyPurple Crab 14789 352 418 6374 45 845 379 Malus Robusta 12715 586 7073901 203 669 326 Makamic Crab 13361 283 411 5379 169 731 294 Profusion13792 208 442 7241 115 672 283 Royalty 26776 944 35 30847 trace 2296 323Liset Crab 17334 22 511 21925 99 182 348 Red Splendor Crab 15934 152 2020906 trace trace 14 Tar Tan 5092 295 116 882 22 168 49 L moine PurpleCrab 10156 136 342 3750 120 570 230 Fuji 2195 168 46 913 18 68 24

[0044] TABLE 3 Total Chlorogenic Total Polyphenol Acid EpicatechinProcyanidin PB1 PB2 PC1 Kind Content (ppm) (ppm) (ppm) Content (ppm)(ppm) (ppm) (ppm) Da Sha Guo 4753 427 113 1308 16 182 61 Hai Tang Hua16017 193 557 10877 133 1020 424 Lomg Dong Hai Tang 6606 77 8 4798 2 135 Qin Guan 2124 217 3 559 2 2 trace Sha Guo 4277 188 37 783 17 56 19Xiang Hong Sha Guo 8159 498 189 4129 30 366 156 Xiao Huang Hai Tang 7383188 92 4654 14 173 67 Yan Tai Sha Guo 6231 263 89 2963 20 152 48 Yin GyeHai Tang 13243 569 321 7309 14 577 250 Yarlington Mill 7157 1039 4521622 166 322 100 Aldenham Purple Crab 9161 129 456 7267 162 619 271 OlgaCrab 5104 176 144 3224 54 266 96

[0045] As shown in Table 2, the crab apples have a higher totalpolyphenol content than Fuji which is an apple to be eaten fresh. It isobvious that the Adams Crab and Royalty species contain about 10 timesas much polyphenol as Fuji. Further, in terms of the components of thepolyphenol, it is obvious that most of the crab apples containchlorogenic acid and epicatechin in large quantity. In addition, it hasbeen disclosed that most of the crab apples contain a large quantity ofcondensed tannins which are responsible for many functionalities of theapple fruit polyphenol. It has also been disclosed that of the condensedtannins, the contents of dimer fraction procyanidins B1 (PB1) and B2(PB2) and a trimer fraction procyanidin C1 (PC1) which are known to havea hair-growing effect as disclosed in JP-A-8-503021 are also very highin most of the crab apples. Further, it is also obvious that by use ofRoyalty containing little procyanidin B1 (PB1), a purified product ofthe procyanidin B2 (PB2) can be obtained easily. As for Red SplendorCrab, the total procyanidin content is high, but neither PB1 nor PB2 isdetected, and the content of PC1 is also low. Therefore, it can be saidthat this species is an excellent material for purifying procyanidinfractions (multimers larger than a trimer) other than PB1, PB2 and PC1.

[0046] Further, as shown in Table 3, Hai Tang Hua contains at least 10times as much procyanidins as Fuji. The species also has high contentsof PB1, PB2 and PC1. Yin Gye Hai Tang contains PB2 in much largerquantity than PB1, since both PB1 and PB2 are a dimer, it is difficultto separate them, and yields thereof are low. However, by use of thespecies as a raw material, PB2 can be purified easily. On the otherhand, Yarlington Mill is excellent as a raw material from which PB1 canbe separated and purified easily. Similarly, although Lomg Dong Hai Tanghas a high total procyanidin content, it has low contents of PB1, PB2and PC1. It can be said that this species is an excellent raw materialto purify procyanidin fractions (multimers larger than a trimer) otherthan PB1, PB2 and PC1.

EXAMPLE 3

[0047] (Analysis of Polyphenol in Juice of Crab Apple from Uyghur inChina)

[0048] Apples produced in the Uyghur Province in China were collected,and polyphenol components in crab apple juices prepared by the samesample preparation process as used in the above Example 1 except that600 ppm of sulfurous acid was added to Uyghur crab apples 1 and 2 and350 ppm of sulfurous acid was added to an Uyghur crab apple 3 weremeasured. A total polyphenol content was determined by measuringabsorbance at 280 nm and also expressed in terms of chlorogenic acid.Other components were quantified in the same manner as in the aboveExample 2. The results are shown in Table 4. TABLE 4 Total TotalPolyphenol Chlorogenic Procyanidin Content Acid Epicatechin Content PB1PB2 PC1 Kind (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) Uyghur 16418 15911150 17376 203 1161 558 Crab Apple 1 Uyghur 13209 1254 1130 16368 2451222 454 Crab Apple 2 Uyghur 11770 1232 1034 13691 197 1107 407 CrabApple 3

[0049] The Uyghur Crab Apples 1, 2 and 3 shown in Table 4 are crabapples produced in I-rei in the Uyghur Province. These apples are onesgrown in an old growth forest of crab apples, and their species are notidentified. All these crab apples contain a large quantity ofpolyphenol. Particularly, the contents of PB1 and PB2 which are aprocyanidin dimer and the content of PC1 which is a trimer are veryhigh. While a value obtained by dividing the total content of PB1, PB2and PC1 by the total polyphenol content is 5% for Fuji, the value is11.7% for the crab apple 1, 14.5% for the crab apple 2, and 14.5% forthe crab apple 3. Since PB1, PB2 and PC1 have a hair-growing effect, itis understood that it is preferable to use these crab apples as rawmaterials so as to purify a fraction containing these 3 components.

EXAMPLE 4

[0050] (Production of Polyphenol Fraction from Crab Apple Juice)

[0051] By use of a ripe crab apple produced in the Uyghur Province inChina in 1999 as a raw material, a juice was prepared by the same samplepreparation process as used in the above Example 1. 190 ml of theobtained juice was passed through a Sepabeads SP-850 resin columnmanufactured by Mitsubishi Chemical Corporation at room temperature, andthe column was then washed with deionized water whose amount was threetimes as large as the volume of the column. After washing, a 50% ethanolaqueous solution whose amount was three times as large as the volume ofthe column was passed through the column so as to elute and recover apolyphenol from the resin. As a result of freeze-drying the obtained 50%ethanol fraction, 858 mg of polyphenol powder was obtained. As a resultof quantifying polyphenol components in this powder by the same processas used in the above Example 2, it was found that in addition to 1.1% ofcatechin and 14.0% of epicatechin, 1.5% of procyanidin B1, 15.9% ofprocyanidin B2 and 6.8% of procyanidin C1 which are condensed tanninsare contained. It was proved that a product containing a highconcentration of polyphenol can be produced from an crab apple easily.

[0052] Possibility of Industrial Utilization

[0053] As described above, according to the present invention, by use ofa ripe crab apple as a raw material and a specific purification process,a fruit polyphenol rich in such components as condensed tannins(procyanidins), chlorogenic acid and epicatechin can be providedeconomically and efficiently.

1. A fruit polyphenol obtained by subjecting a crab apple fruit topressing and/or extraction.
 2. The fruit polyphenol of claim 1, whereinthe crab apple is at least one selected from the group consisting of HuBei Hai Tang, Yin Gui Hai Tang, Xi Fu Hai Tang, Zhong Guo Ping Guo, CuiGuo, Ya Hai Tang, Ya Sha Guo, Ya Ping Guo, Adams Crab, Geneva, Gorgeous,Jay Darling, Eleyi, Eley Purple Crab, Malus Robusta, Makamic Crab,Profusion, Royalty, Liset Crab, Red Splendor Crab, Tar Tan, and LemoinePurple Crab.
 3. The fruit polyphenol of claim 1, wherein the crab appleis an apple produced in the Uyghur Province in China, Kazakhstan orUzbekistan.
 4. A process for producing a fruit polyphenol whichcomprises the steps of subjecting a crab apple fruit to pressing and/orextraction and then purifying the obtained juice and/or extract so as toobtain a polyphenol fraction.
 5. The process of claim 4, wherein 350 to3,000 ppm of sulfurous acid is added at the time of pressing the crabapple fruit.
 6. The process of claim 4, wherein at least 100 ppm ofsulfurous acid is added when a proper amount of alcohol is mixed intothe crab apple fruit which is then crushed at the time of subjecting thecrab apple fruit to extraction, and at least 50 ppm of sulfurous acid isadded after preparation of a clear extract.
 7. The process of claim 4,wherein the crab apple is at least one selected from the groupconsisting of Hu Bei Hai Tang, Yin Gui Hai Tang, Xi Fu Hai Tang, ZhongGuo Ping Guo, Cui Guo, Ya Hai Tang, Ya Sha Guo, Ya Ping Guo, Adams Crab,Geneva, Gorgeous, Jay Darling, Eleyi, Eley Purple Crab, Malus Robusta,Makamic Crab, Profusion, Royalty, Liset Crab, Red Splendor Crab, TarTan, and Lemoine Purple Crab.
 8. The process of claim 4, wherein thecrab apple is an apple produced in the Uyghur Province in China,Kazakhstan or Uzbekistan.